Journal of Medical Virology 87:1276–1284 (2015)

The Impact of Viral Dynamics on the Clinical Severity of Infants With Respiratory Syncytial Virus Bronchiolitis Lili Zhou,1,2,3 Qiuyan Xiao,1,2 Yao Zhao,1,2 Ailong Huang,4 Luo Ren,1,2 and Enmei Liu5* 1

Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, China 2 Key Laboratory of Pediatrics in Chongqing CSTC2009CA5002, Chongqing, China 3 Department of Respiratory Medicine, Women and Children’s hospital, Ganzhou, Jiangxi, China 4 Key Laboratory of Molecular Biology of Infectious Diseases, Ministry of Education, Chongqing Medical University, Chongqing, China 5 Department of Respiratory Medicine, Children’s Hospital, Chongqing Medical University, Chongqing, China

The impact of dynamic respiratory syncytial virus (RSV) load on the clinical severity of hospitalized infants with bronchiolitis has not been clarified. Nasopharyngeal aspirates were obtained from 60 infants who were diagnosed with bronchiolitis within 96 hr of wheezing onset upon admission and on days 3, 5, and 7 in the hospital, and 17 respiratory viruses were detected. The RSV load was quantified by real-time qPCR for RSV subtypes A and B at different time points. Scoring criteria were used to evaluate the degree of severity. A total of 40 infants were determined to be RSV-positive, nine were identified as RSV subtype A (RSVA), and 31 were RSV subtype B (RSVB). The peak RSV load was observed upon admission, and the RSV load decreased significantly over time; in addition, this decrease began to have significant differences on day 5. There was a positive correlation between the RSV load and the clinical score (r2 ¼ 0.121 and P < 0.001). According to the clinical scores, the infants in the severe group tended to have higher RSV loads than those in the moderate and mild groups. Multivariate logistic regression models revealed that the viral load on day 3 was independently associated with the degree of severity. This study elucidated that a higher mean RSV load was associated with a more severe disease and a longer duration of hospitalization and symptoms. This study also clarified RSV replication in infants and provides a theoretical basis for specifying an anti-RSV therapy strategy. J. Med. Virol. 87:1276–1284, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:

RSV load; clinical severity; time points; infants; bronchiolitis


INTRODUCTION Bronchiolitis is a common acute respiratory tract infection, which is a distressing, potentially life-threatening respiratory condition that affects young babies [Rosalind, 2006]. It is generally characterized by acute inflammation, edema, and necrosis of epithelial cells lining small airways, leading to rhinorrhea, cough, wheezing, hyperexpansion of the lungs, hypoxia, and respiratory distress [Panitch, 2003; Pavia, 2011]. RSV is the most common etiological agent of bronchiolitis. It was estimated that 66,000–199,000 children younger than 5 years died from RSV-associated acute respiratory tract infection in 2005, with 99% of these deaths occurring in developing countries [Nair et al., 2010]. In the UK, the RSV-attributed death rate in infants has

Abbreviations: RSV, respiratory syncytial virus; RSVA, respiratory syncytial virus subgroup A; RSVB, respiratory syncytial virus subgroup B; IVA, influenza virus A; IVB, influenza virus B; IVC, influenza virus C; CoV-229E, human coronaviruses CoV-229E; CoV-OC43, human coronaviruses CoV-OC43; hMPV, human metapneumovirus; PIV1-4, parainfluenza virus 1-4; hRVA, human rhinovirus subtypes A; hRVB, human rhinovirus subtypes B; hRVC, human rhinovirus subtypes C; hBoV, human Bocavirus; AdV, adenovirus Grant sponsor: China Special Grant for the Prevention and Control of Infection Diseases; Grant number: 2012zx10004212.; Grant sponsor: National Key Specialty; Grant number: [2011] 873. Conflict of interest: None.  Correspondence to: Enmei Liu, Department of Respiratory Medicine, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, China. E-mail: [email protected] Accepted 28 October 2014 DOI 10.1002/jmv.24111 Published online 23 April 2015 in Wiley Online Library (

Dynamic RSV Load and Clinical Severity


been estimated to be 8.4 per 100,000 [Fleming et al., 2005]. The incidence and mortality can vary substantially from year to year. At present, there is no special medicine for the treatment of bronchiolitis. It has been reported that approximately 1% of infants with bronchiolitis need to go to the hospital for treatment and that 10% of them require treatment in an intensive care unit (ICU) [Nagakumar and Doull, 2012]. It has been reported that the RSV loads after symptom onset are correlated with disease severity in hospitalized infants [Buckingham et al., 2000; Scagnolari et al., 2012]. Higher RSV loads predict disease severity in infants who were previously healthy correlating with longer durations of hospitalization [DeVincenzo et al., 2005; El Saleeby et al., 2011]. Recent studies also report that disease severity is positively correlated with the viral load during primary RSV infection [Houben et al., 2010]. Some reports have investigated the relationship between RSV viral dynamics and disease severity. In their study, the viral loads on day 3 were associated more significantly with a requirement for intensive care and respiratory failure than the viral loads at earlier time points. Faster RSV clearance was independently associated with shorter hospitalization [El Saleeby et al., 2011]. Clinical improvement was associated with a reduction of viral quantity after three days of hospitalization [Jansen et al., 2010]. However, the relationship between changes in the RSV load dynamics and the disease severity of RSV-infected infants has not been studied in detail. Understanding the relationship between the clinical severity of bronchiolitis and the dynamic RSV load will aid the risk factor assessment of clinical severity and help the development of timely intervention therapy. It will thus provide a conceptual background for understanding the disease pathogenesis of RSV and other respiratory virus infections. This study selected the study subjects with strict inclusion criteria, collected nasopharyngeal aspirates specimens from patients at different time points and used clinical scores to evaluate the bronchiolitis severity objectively to confirm correlations between the severity and changes in the RSV load dynamics during the course of bronchiolitis.

MATERIALS AND METHODS Study Patients Inclusion criteria: previously healthy children aged 24 months diagnosed with bronchiolitis within 96 hr of wheezing onset and hospitalized at the Children’s Hospital of Chongqing Medical University were enrolled in this prospective study during two successive winter seasons (2012–2014). The diagnosis of bronchiolitis is according to the Zhu Futang textbook of Pediatrics [Zhang, 2002]. Bronchiolitis was diagnosed from the presence of a history of upper respiratory tract infection followed by acute onset of respiratory distress with cough, tachypnea, retraction, wheezing, and diffuse crackles on auscultation. Patients were excluded if they had received antiviral (ribavirin) and immunomodulatory agents (prednisone or hydrocortisone) in the preceding two weeks. Children with wheezing, breathlessness, and obstruction of the airways, in whom similar episodes had previously been diagnosed and treated by a physician, were diagnosed as recurrent wheezing and were excluded. Qualified pediatricians identified bronchiolitis and its clinical severity and collected clinical information confidentially. The demographic data and clinical history were collected from guardians via interviews. This study was approved by the Ethics and Research Council of Chongqing Children’s Hospital, and signed consent was obtained from each child’s parents or foster parents. Disease Severity Scoring Criteria Scoring criteria was used to evaluate the severity of bronchiolitis [Wang et al., 1992; Luo et al., 2010]. Based on the total score, the disease severity was divided into the following ranks: mild, 0–4.9; moderate, 5–8.9; and severe, 9–12 (details in Table I). Specimens and Virus Detection Nasopharyngeal aspirates were collected by a standardized technique from patients upon admission and on days 3, 5, and 7 in the hospital. For samples, the nasopharyngeal aspirates were collected by welltrained nurses during the same time of day. A disposable controllable suction tube with the negative

TABLE I. Clinical Scores Used to Estimate the Degree of Clinical Disease Severity*

Respiratory rate (breaths/min) Wheezing Retractions General condition

0 point

1 point

2 point

3 point






terminal expiratory or heard only with a stethoscope intercostal only _

entire expiration or audible on expiration without a stethoscope tracheosternal _

inspiration and expiration without a stethoscope severe with nasal flaring irritable, lethargic, poor feeding

none normal


The disease severity was evaluated using the clinical score index described by Wang et al. [Wang et al., 1992].

J. Med. Virol. DOI 10.1002/jmv


Zhou et al.

pressure method was used to collect the nasopharyngeal aspirates through the following operation: a syringe was connected to the plastic disposable sputum suction tube; the sputum tube was placed carefully and gently into the nasopharynx of patients; the saline injection was gently pushed; the disposable controllable suction tube, which is connected to the negative pressure device, can then be used to suck as much as possible of the nasopharynx secretion; and each specimen was diluted with 2 ml of physiological saline. The specimens were maintained at 4˚C for a maximum of 4 hr and then stored at 80˚C until further processing. The viral DNA and RNA were extracted from 200 ml aliquots of the nasopharyngeal aspirates samples using the QIAamp MinElute Virus Spin kit (Qiagen, Hilden, Germany). RNA was applied as the template for cDNA synthesis using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). The DNA and RNA extractions and cDNA products were used for the subsequent testing of 17 respiratory viruses. The 17 respiratory viruses were detected as follows [Coiras et al., 2003; Coiras et al., 2004; Lu et al., 2006; Granados et al., 2012]: nested PCR assays were used to detect human RSVA, RSVB, IVA, IVB, IVC, CoV229E, CoV-OC43, hMPV, PIV 1–4, and AdV. Realtime qPCR was used to detect hRV and hBoV. Real-Time qPCR for RSV Subtypes A and B For the RSV-positive samples tested by nested PCR assays, the RSV load was determined by real-time qPCR. The primers and probe for RSVA and RSVB N gene amplification are listed in Table II [Hu et al., 2003; Van-Woensel et al., 2003]. The sequence of the N gene for the RSV A2 strain (GenBank accession number M11486) and that for the RSV B strain (GenBank accession number D00736) were used for the design of the primers and probes. The plasmidamplified target fragment was cloned into the pMD19-T vector (TaKaRa Biotechnology, Dalian, China) and verified by sequencing. The plasmid DNA concentrations were detected with an ND-1000 spectrophotometer (Nanodrop). Real-time qPCR was carried out in a total reaction volume of 20 ml consisting of 10 ml of TaqMan Universal Master Mix (Applied Biosystems), 0.6 ml (10 mM) of each primer, 0.6 ml

TABLE II. Primers and Probes for RSVA and RSVB N Gene Amplification Primer and Probe RSVA-F RSVA-R RSVA-P RSVB-F RSVB-R RSVB-P


J. Med. Virol. DOI 10.1002/jmv

(10 mM) of the probe, 2 ml of template and 6.2 ml of double-distilled water. The real-time qPCR thermal cycling reaction and quantitative measurement were performed in a 96-well format in a StepOne RealTime PCR instrument (Applied Biosystems) using the following conditions: one cycle at 50˚C for 2 min, one cycle at 95˚C for 10 min, 40 cycles at 95˚C for 15 sec, and one cycle at 60˚C for 1 min. Strict laboratory procedures were followed to prevent PCR contamination, including separate spaces for sample handling, the Master Mix ingredients, and the plasmid templates. All of the runs included negative controls and plasmid in each PCR assay. Representative results from the real-time qPCR quantification were found for the serial dilutions of RSVA and RSVB plasmids (101–107 copies/reaction) with the baseline-corrected fluorescence plotted against the cycle number. The standard curves were positioned within each plate, and each run was performed in duplicate. For standard curves of five independent runs, the mean correlation coefficient of RSVA was 0.996 with a standard deviation of 0.004, and the mean value of the slope was 3.53 with a standard deviation of 0.04. The mean correlation coefficient of RSVB was 0.998 with a standard deviation of 0.02, and the mean value of the slope was 3.56 with a standard deviation of 0.17. The standard curves are shown in Figure 1. The RSV load values are expressed as log10 copy number of RSV-RNA/ml in nasopharyngeal aspirates. Statistical Analyses The data were analyzed using the SPSS 17.0 software package. GraphPad Prism5 (GraphPad Software Inc.) was used to generate figures and graphs. The categorical variables were compared using the Chi-square test, and continuous variables were compared using Student’s t-test. A two-sided P < 0.05 was considered statistically significant. A correlation analysis of the RSV load and clinical severity data was conducted. Logistic regression was used to determine whether the viral load is independently associated with the degree of severity. RESULTS Demographic Clinical and Virological Characteristics A total of 60 infants met the inclusion criteria. RSV was detected in 40 of these infants (31 were RSVB and 9 were RSVA), six of the infants were found to have other respiratory viruses, and no viruses was detected in 14 children. Single RSV infection was detected in 32 (80%) infants, whereas co-detections with other respiratory viruses were detected in eight (20%) infants. The demographic, clinical, and virological characteristics of infants with RSV are shown in Table III. The median age of the infants was four (range, 1–11) months. Among the

Dynamic RSV Load and Clinical Severity


Fig. 1. Representative results from the real-time qPCR quantification of serial dilutions of RSVA and RSVB plasmids (101–107copies/reaction). The baseline-corrected fluorescence was plotted against the cycle number. A: Standard curve and amplification plot of RSVA. B: Standard curve and amplification plot of RSVB.

infants, 28 were boys, and 12 were girls. Eighteen (45%) infants had severe bronchiolitis (clinical score 9), 18 (45%) had moderate bronchiolitis (clinical score between 5 and 8.9), and four (10%) infants had mild bronchiolitis (clinical score

The impact of viral dynamics on the clinical severity of infants with respiratory syncytial virus bronchiolitis.

The impact of dynamic respiratory syncytial virus (RSV) load on the clinical severity of hospitalized infants with bronchiolitis has not been clarifie...
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